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Celgene Corporation provided 5AC. lines were treated with 5AC and MGC. Real-time polymerase chain reaction (PCR) and Western blotting were performed to assess MAGE-A3 ON-01910 (rigosertib) RNA and protein levels, respectively. Chromium-release assays and interferon (IFN) secretion assays were employed to ascertain MAGE-A3 CTL specificity against treated targets. == Results == Gene expression analysis revealed that MAGE-A3 is expressed in MM patients at diagnosis (25%) and at relapse (49%). We observedde novoexpression of MAGE-A3 RNA and protein in MAGE-A3-negative cell lines treated with 5AC. MGC treatment alone did not induce expression but sequential 5AC/MGC treatment led to enhanced expression and augmented recognition by MAGE-A3-specific CTL, as assessed by51Cr-release assays (P= 0.047) and enzyme-linked immunosorbent assay (ELISA) for IFN- secretion (P= 0.004). == Conclusions == MAGE-A3 is an attractive target for immunotherapy of MM and epigenetic modulation by 5AC, and MGC can induce MAGE-A3 expression and facilitate killing by MAGE-A3-specific CTL. Keywords:5-azacitidine, cancer-testis antigen, demethylation, epigenetics, histone deactylase inhibitor, hypomethylation, MAGE-A3, MGCD0103, multiple myeloma == Introduction == Currently available therapies offer poor long-term outcomes for multiple myeloma (MM) patients with a high-risk genetic signature (1). This group accounts for 15% of newly diagnosed patients and 75% of those with relapsed disease. Approaches to intensify therapies in this group of patients have led to cumulative toxicity and host exhaustion and have not improved overall survival (OS). Therefore, new therapeutic approaches that are both non-toxic and non-cross-resistant with chemotherapy are desperately needed for these patients. One potential answer lies in immune therapies targeting tumor-specific antigens, which may eradicate chemoresistant tumor-cell clones without inducing significant toxicities. For example, vaccination with tumor-specific antigens and transfer of tumor-specific T cells is safe and has induced clinical responses in lung cancer and melanoma (2,3). Targets of particular interest are the cancer-testis antigens (CT-Ag), whose expression in normal tissues is restricted to immunoprivileged sites such as the testes, ensuring that immune responses generated toward these antigens will be non-toxic to normal tissues. CT-Ag expression is common in MM (48) and has been linked to poor prognosis (9,10). CT-Ag expression in cancer is probably the result of global hypomethylation, specifically of CpG islands at promoter sites (1113).This phenomenon may also explain reports of coordinate expression of ON-01910 (rigosertib) multiple CT-Ag in malignancies, including MM (8,1416). One potential concern in targeting CT-Ag for immunotherapy is that CT-Ag-negative clones could lead to tumor escape (1720). Interestingly,de novoinduction of CT-Ag expression has been achieved with hypomethylating agents such as 5-aza 2-deoxy-cytidine (DAC) and its nucleoside analog 5-azacitidine (5AC) (2027), which incorporates into RNA and, to a lesser extent, DNA (12,28). 5AC has been approved by the food and drug administration (FDA) for use in myelodysplastic syndrome (MDS). Phase I and II clinical trials investigating the use of 5AC in MM have been initiated (protocol IDNCT00761722andNCT00412919). Further increases in hypomethylating agent-induced gene expression have been achieved with histone deactylase inhibitors (HDACi) such as trichostatin A, valproic acid and MGCD0103 (MGC) via hyperacetylation of the histone core (12,29,30). We have reported previously that potent immune responses to the CT-Ag MAGE-A3 can be induced by vaccination of a MM patient with MAGE-A3-positive disease with MAGE-A3 recombinant protein (31). We therefore wished to study whether the combination of demethylating agents and HDACi could optimize such therapy. We first analyzed expression of the CT-Ag MAGE-A3 in MM patients and correlated expression with validated disease subgroups identified by gene-expression profiling (GEP) (32) and survival. We then studied whether 5AC induced expression of MAGE-A3 in MM cells and whether any up-regulation could be enhanced with MGC. Finally, we assessed whether MAGE-A3/HLA-A*6801 -specific cytotoxic T lymphocytes (CTL) could kill 5AC/MGC-treated targets. == Methods == == Subject samples, GEP, subgroup and survival curve analyses == Normal tissue RNA (plasma cell, lung, uterus, kidney, stomach, brain, breast, spleen, prostate, skeletal muscle, testis, thymus, liver, ovary, heart and small intestine) were obtained from Clontech (Mountain View, CA, USA). RaLP Bone marrow was collected from healthy donors and patients with MM, after informed consent, in accordance with the Declaration of Helsinki. Approval was obtained from the University of Arkansas for Medical Sciences (UAMS, Little Rock, AR, USA) Institutional Review Board for sample procurement. CD 138-positive plasma cells were purified using CD 138 antibody (Ab)-coated magnetic beads (Miltenyi Biotec Inc., Auburn, CA, ON-01910 (rigosertib) USA), as described previously (33). GEP (33), molecular subgroup (32), high-risk group classifications (34) and survival curve (32) analyses ON-01910 (rigosertib) were performed as reported previously using samples obtained.